The present invention relates to a novel process for producing pyrrole-2-carboxylic acid from pyrrole by action of microorganisms. Pyrrole-2-carboxylic acid and its derivatives are useful as raw materials for various drugs.
In vivo enzymatic decarboxylation for amino acids and xcex1-keto acids has been studied well, so that the properties of enzymes involved in the reaction is now clearly understood. However, much remains unclear regarding nonoxidative decarboxylase for aromatic carboxylic acids.
A known nonoxidative decarboxylating reaction of aromatic carboxylic acid by a microorganism is A reaction to convert a hydroxybenzoic acid into phenol (Microb. Ecol. , 20, 103, 1990). Moreover, bacteria belonging to the genus Citrobacter are known to decarboxylate a gallic acid so as to generate and accumulate pyrogallol (Agric. Biol. Chem., 46, 2539, 1982).
On the other hand, a microorganism known to catalyze carbon dioxide fixation to aromatic compounds is a bacterium belonging to the genus Pseudomonas that converts phenol to 4-hydroxybenzoic acid in the first step towards decomposition of phenol (Arch. Microbiol., 148, 213, 1987).
Furthermore, properties of an enzyme derived from the genus Clostridium have been examined in detail. That is, the enzyme has been shown to catalyze decarboxylation and carbon dioxide fixation of 4-hydroxybenzoic acid and of 3,4-dihydroxybenzoic acid by a reversible reaction (Appl. Environ. Microbiol., 60, 4182, 1994).
Industrial application of the synthesis of carboxylic acid by carbon dioxide fixation to aromatic compounds has great value. However, carbon dioxide-fixing activity of the above-mentioned microorganisms and enzyme is so poor that they cannot easily be practically applied.
As a result of thorough studies on enzymes which catalyze carbon dioxide fixation to aromatic compounds, we have previously found that pyrrole-2-carboxylic acid decarboxylase derived from the genus Bacillus performs carbon dioxide fixation to pyrrole, so as to synthesize pyrrole-2-carboxylic acid (Eur. J. Biochem., 253, 480, 1998).
However, the reaction system of this enzyme requires the co-existence of an organic acid, such as ammonium acetate. This is inconvenient when considering, for example, the isolation of products.
Under the circumstances, we have completed the present invention as a result of devoted study, by finding that microorganisms, which are derived from bacteria belonging to the genus Serratia and decarboxylate pyrrole-2-carboxylic acid, stably perform the reaction in the absence of organic acid ammonium.
That is, the present invention provides a process for producing pyrrole-2-carboxylic acid comprising the steps of allowing a microorganism, which is derived from the genus Serratia, is capable of catalyzing decarboxylation of pyrrole-2-carboxylic acid, and is capable of catalyzing the synthesis of pyrrole-2-carboxylic acid from pyrrole in the presence of carbonate ion, or the microorganism optionally processed, to act on pyrrole; and recovering the pyrrole-2-carboxylic acid which is generated.
Further, the present invention provides the above process for producing pyrrole-2-carboxylic acid wherein the microorganism or the microorganism optionally processed is allowed to act in the absence of organic acid.
Furthermore, the present invention provides an enzyme which is extracted from a microorganism belonging to the genus Serratia, is capable of catalyzing decarboxylation of pyrrole-2-carboxylic acid, and is capable of catalyzing the synthesis of pyrrole-2-carboxylic acid from pyrrole in the presence of carbonate ion.
The present invention also provides the above described enzyme wherein the microorganism belonging to the genus Serratia is Serratia grimesii (IF013537), Serratia marcescens (IAM12142), or Serratia rubidaea (IF012973).
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 11-539, which is a priority document of the present application.
Now a detailed description of this invention will be given.
A microorganism of the present invention is derived from the genus Serratia, catalyzes decarboxylation of pyrrole-2-carboxylic acid, and has activity to convert pyrrole to pyrrole-2-carboxylic acid in the presence of carbonate ion. Examples of such a strain include Serratia grimesii (IF013537), Serratia marcescens (IAM12142), and Serratia rubidaea (IF012973).
These microorganisms are known. Strains IFO13537 and IFO12973 are easily obtained from the Institute for Fermentation, 17-85, Juso-honmachi, 2-chome, Yodogawa-ku, Osaka 532-8686, Japan, and IAM12142 from the Institute of Molecular and Cellular Biosciences (IAM), University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan, respectively.
Generally, a medium for culturing microorganisms in the present invention may be any one in/on which they can grow. Examples of carbon sources include sugars, such as glucose, sucrose, and maltose; organic acids, such as acetic acid, citric acid and fumaric acid, or salts thereof; and alcohols, such as ethanol and glycerol. Examples of nitrogen sources include general and natural nitrogen sources, such as peptone, meat extract, yeast extract, and amino acid, and various inorganic and organic acid ammonium salts and the like. If necessary, inorganic salts, trace mineral salts, vitamins and the like are added appropriately. Moreover, addition to a medium of pyrrole-2-carboxylic acid, benzoic acid, anthranilic acid and their derivatives and the like as inducers for enzyme production is effective to obtain high enzymatic activity.
Culturing may be performed by standard techniques. For example, culturing is performed at pH 4 to 10, and at a temperature ranging from 15xc2x0 C. to 40xc2x0 C. aerobically for 6 to 96 hours.
The reaction for producing pyrrole-2-carboxylic acid can be performed by allowing the microorganisms above or those optionally processed to contact with pyrrole in the presence of carbonate ion and in water or in a buffer such as phosphoric acid buffer, carbonic acid buffer, boric acid buffer and the like.
Examples of an optionally processed microorganism include, but are not limited to, a culture product from the microorganisms, viable cells, dried cells, disrupted cells, enzyme extract solution, and crude/purified enzyme.
Reaction may be performed generally at pH ranging from 4 to 10, preferably pH 5 to 9, and at a temperature ranging from 0 to 60xc2x0 C., preferably 5 to 50xc2x0 C. Suitable concentration of pyrrole varies depending on reaction temperature and pH. Generally, a preferable concentration of pyrrole ranges from 50 to 300 mM. In addition, the amount of microorganisms or that of those optionally processed to be used generally ranges from 0.01 to 5 wt % on a conversion with dried cell relative to a substrate.
Examples of the sources of carbonate ion include carbonates, such as sodium, potassium, and ammonium carbonates, of which ammonium bicarbonate, sodium bicarbonate and the like are particularly effective. Since this reaction is an equilibrium reaction, reaction yield can be further increased by performing the reaction under carbon dioxide gas pressure in addition to the presence of these salts.
Pyrrole-2-carboxylic acid can be recovered from the reaction solution by known methods including bacteria elimination, concentration, anion exchange chromatography, and crystallization.